Enhancement of B-MYB transcriptional activity by ZPR9, a novel zinc finger protein.

By using the yeast two-hybrid system, the zinc finger protein ZPR9 was identified as one of the B-MYB interacting proteins that associates with the carboxyl-terminal conserved region of B-MYB. ZPR9 was found to form in vivo complexes with B-MYB, as demonstrated by in vivo binding assay and coimmunoprecipitation experiments of the endogenously and exogenously expressed proteins. Deletion analysis revealed that this binding was mediated by all three functional domains, an amino-terminal DNA-binding domain, a transactivation domain, and a carboxyl-terminal conserved region of B-MYB. We show that the interaction of ZPR9 with B-MYB is functional because cotransfection of ZPR9 significantly up-regulates B-MYB transcriptional activity in a dose-dependent manner. In addition, coexpression of ZPR9 with B-MYB caused the accumulation of B-MYB, as well as ZPR9, in the nucleus. Furthermore, constitutive expression of ZPR9 in human neuroblastoma cells induces apoptosis in the presence of retinoic acid. These results strongly suggest that ZPR9 plays an important role in modulation of the transactivation by B-MYB and cellular growth of neuroblastoma cells.

B-MYB is a member of the MYB family of transcription factors, which is ubiquitously expressed and is involved in controlling cell proliferation and differentiation (1)(2)(3)(4)(5). B-MYB is phylogenetically the most divergent among the three MYB proteins, A-MYB, B-MYB, and c-MYB (6). Recent reports showed that the CDK2-cyclin A complex could induce phosphorylation of B-MYB and potentiate the B-MYB transactivating function and that this activation was also induced by truncation of the carboxyl terminus of B-MYB, suggesting that posttranslational modifications are required for relieving the constitutive repression of B-MYB (7)(8)(9)(10). In addition, a recent study (11) indicated that the B-MYB transactivation correlates with the binding of some cofactors to the carboxyl-terminal conserved region, suggested as a protein binding domain and a putative phosphorylation site.
The MYB proteins are composed of three functional domains for transactivation, an amino-terminal DNA-binding domain, a central acidic region (transactivation domain), and a carboxyl-terminal negative regulatory domain containing the leucine zipper motif (12). Recently, all these domains have been reported to be involved in interactions with several cellular proteins. The DNA-binding domain of c-MYB was found to bind with several proteins such as p100 coactivator (13,14), c-Maf transcription factor (15), Cyp-40 peptidylprolyl isomerase (16), HSF3 (17), nucleolin (18), and retinoic acid receptor (19). In addition, recent reports have shown that the DNA-binding domain of A-MYB and B-MYB interacts with several nuclear proteins (18,20) and poly(ADP-ribose) polymerase (PARP), 1 which is associated with chromatin (21), respectively. On the other hand, the cAMP-response element-binding protein has been demonstrated to interact directly with the transactivation domain of both c-MYB and A-MYB and potentiate their transcriptional activity (22,23). The leucine zipper motif of the carboxyl-terminal domain was also found to associate with several proteins, including p26/28 (24), p67, and p160 (25,26), and ATBF1 transcription factor (27), but these interactions except for ATBF1 have not been implicated in the regulation of MYB function so far. From these results, it is tempting to speculate that additional proteins may be involved in the regulation of transactivation by B-MYB, probably by association with the carboxyl-terminal conserved region that shows significant homology with other members of the MYB gene family such as A-MYB and c-MYB.
ZPR9, a zinc finger protein, was originally identified as a novel cellular partner for the MPK38 serine/threonine kinase that may be involved in early T cell activation by concanavalin A (28) and embryonic development (29,30). ZPR9 is a 52-kDa protein containing three zinc finger motifs and a physiological substrate of MPK38 kinase in vivo (31).
Here we show that ZPR9 binds to B-MYB in vivo and that the overexpression of ZPR9 induces apoptosis, instead of neural differentiation, in the neuroblastoma cells treated with retinoic acid. Binding of ZPR9 to B-MYB can stimulate the B-MYB transcriptional activity. In addition, we provide evidence that all three functional domains of B-MYB physically interact with ZPR9 in vivo. We also demonstrate that the coexpression of ZPR9 with B-MYB causes the accumulation of both ZPR9 and B-MYB in the nucleus.

EXPERIMENTAL PROCEDURES
Reagents-The eukaryotic glutathione S-transferase (GST) expression vector (pEBG) and pFLAG-CMV-2 vector with a FLAG epitope were obtained as described previously (32). The anti-GST antibody was as described (32). The pT81luc 3xA reporter plasmid (33), containing three copies of the "A box" Myb-binding sites from the nim promoter, was a kind gift from Dr. Scott A. Ness (the University of New Mexico, Albuquerque, NM). The expression vector pCEV27 was kindly provided by Dr. D-Y. Shin (Danguk University, Chonan, Korea). The anti-FLAG (M2) antibody, all-trans-retinoic acid (RA), BisBenzimide (H 33258), isopropyl-␤-D-thiogalactopyranoside, dithiothreitol, aprotinin, and phenylmethylsulfonyl fluoride were purchased from Sigma. Polyvinylidene difluoride membrane was obtained from Millipore Corp.
[␥-32 P]ATP was purchased from PerkinElmer Life Sciences. The human B-MYB antibody (C-20) raised against the carboxyl terminus was used for immunoprecipitation and Western analysis (Santa Cruz Biotechnology). Oligonucleotides were synthesized from Bioneer Corp. (Cheongwon, Chungbuk, Korea).
Cell Culture-The human neuroblastoma cell line SK-N-BE (2)C and 293T cells, a derivative of human kidney embryonal fibroblast containing SV40 T antigen, were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 100 units/ml penicillinstreptomycin, and 1 mM glutamine as described (34). For cell differentiation experiments, SK-N-BE (2)C cells grown in DMEM supplemented with 10% FBS were plated in 6-well flat-bottomed microplates at a concentration of 4 ϫ 10 5 cells per well the day before retinoic acid (RA) treatment, and the medium was replaced with fresh medium without FBS, containing 5 M all-trans-retinoic acid, every 3 days. The 293T cells were transfected by the calcium phosphate precipitation method as described previously (34).
Plasmid Constructions-The pEBG-B-MYB, an amino-terminally truncated version containing part of the acidic region and a complete conserved region, and pEBG-WT B-MYB, containing a full-length B-MYB cDNA, have been described previously (32). The deletion constructs, pEBG-B-MYB R1 and pEBG-B-MYB R2, were generated by PCR as described (32). To generate two deletion constructs, pFLAG-DBD and pEBG-TA, we performed a PCR using the full-length B-MYB cDNA as the template. The forward primers for DBD (5Ј-GCGAAT-TCATGTCTCGGCGG-3Ј) and TA (5Ј-GCAAGCTTGAGGACAAGGAC-3Ј) contain an EcoRI and a HindIII site (underlined). The reverse primers for DBD (5Ј-GCGGATCCCTCGAGCTCCAG-3Ј) and TA (5Ј-GCGGATCCCAGGCGGTACTC-3Ј) contain a BamHI site (underlined). The amplified PCR products for deletion mutants were cut with EcoRI plus BamHI and HindIII plus BamHI and cloned into pBluescript KS (Stratagene) to generate the KS-DBD and KS-TA constructs, respectively. The pFLAG-DBD was generated after subcloning of an EcoRI/ BamHI fragment of KS-DBD into the EcoRI/BamHI site of pFLAG-CMV-2. The pEBG-TA was generated by subcloning of a ClaI/NotI fragment from KS-TA into pEBG vector. The identity of all PCR products was confirmed by nucleotide sequencing analysis on both strands with the T7 Sequencing TM kit (Amersham Biosciences). The pEBG-CR, a carboxyl-terminally truncated version containing a DNA-binding domain, a transactivation domain, and a complete conserved region, was made by PCR amplification. The amplified PCR product was cut with HindIII plus BamHI and cloned into pBluescript KS to generate the KS-CR construct. The pEBG-CR was created by subcloning of a ClaI/ NotI fragment from KS-CR into pEBG vector. The pEBG-TA1, a carboxyl-terminally truncated version containing part of the transactivation domain and a complete DNA-binding domain, was constructed in several steps. We first cloned the HindIII/EcoRI fragment of full-length B-MYB into pBluescript KS and digested with ClaI plus NotI and subcloned into pEBG, yielding pEBG-TA1. A full-length ZPR9 cDNA obtained from a human normal keratinocyte cDNA library was cloned into pEBG and pFLAG-CMV-2 to generate the pEBG-ZPR9 and pFLAG-ZPR9, respectively (31). The pCEV27-ZPR9 for expression of human ZPR9 was prepared by subcloning of a BamHI/XhoI fragment from pBacPAK9-ZPR9 into pCEV27 vector. The pBacPAK9-ZPR9 was generated by subcloning of an EcoRI/XbaI fragment from pFLAG-ZPR9 into pBacPAK9 (Clontech). For a confocal microscopy, the GFP-B-MYB was created by subcloning of a KpnI/BamHI fragment from KS-B-MYB into pRSGFP-C1 vector (Clontech).
Yeast Two-hybrid Specificity Test-A fish plasmid, pJG4-5 harboring a carboxyl-terminal conserved region of B-MYB, was transformed back into EGY48 cells along with either the bait plasmid, pEG202 harboring ZPR9, or other several bait plasmids available in our laboratory (32). For selection of proteins interacting with the B-MYB, the plate assays of ␤-galactosidase expression were carried out.
In Vivo Binding Assay-293T cells grown in DMEM supplemented with 10% FBS were plated in 6-well flat-bottomed microplates at a concentration of 2 ϫ 10 5 cells per well the day before transfection. 1-5 g of each plasmid DNA was transfected into 293T cells with a calcium phosphate precipitation method. Forty eight hours after transfection, cells were washed three times with ice-cold phosphate-buffered saline (PBS) and solubilized with 100 l of lysis buffer (20 mM Hepes (pH 7.9), 10 mM EDTA, 0.1 M KCl, and 0.3 M NaCl) containing 0.1% Nonidet P-40, 10 g/ml aprotinin, 10 g/ml leupeptin, 10 mM sodium fluoride, 2 g/ml ␣ 1 -antitrypsin, 2 mM sodium pyrophosphate, 25 mM sodium ␤-glycerophosphate, 1 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride. Detergent-insoluble materials were removed by centrifugation at 13,000 rpm for 15 min at 4°C. Approximately 80 l of the cleared lysates were mixed with 15 l of glutathione-Sepharose beads (Amersham Biosciences) and rotated for 2 h at 4°C. Beads were washed three times with the lysis buffer. The bound proteins were eluted by boiling in SDS sample buffer, subjected to SDS-PAGE, and then transferred to polyvinylidene difluoride membranes. The membranes were probed with an anti-FLAG (M2) antibody and then developed using an ECL detection system (Amersham Biosciences).
Luciferase Reporter Assay-293T cells were transfected according to the calcium phosphate precipitation method. After 48 h, the cells were harvested, and luciferase activity was monitored with a luciferase assay kit (Promega) following the manufacturer's instructions. Light emission was determined with a Berthold luminometer (Microlumat LB96P). The cell extracts containing equal amounts of B-MYB and ZPR9, determined by Western blot analysis, were used for luciferase assay. The values were adjusted with respect to expression levels of a cotransfected ␤-galactosidase reporter control, and experiments were repeated at least three times.
Northern Blot Analysis-Total cytoplasmic RNAs were prepared from cells using RNAzol TM B (Biotex Laboratories) as described (28). Approximately 30 g/ml total RNA was electrophoresed through 1.2% agarose-formaldehyde gel and transferred to GeneScreen Plus TM nylon membrane (PerkinElmer Life Sciences). The membranes were hybridized with a 32 P-labeled ZPR9 cDNA probe at 42°C overnight in 50% formamide, 10% dextran sulfate, 7% SDS, 0.25 M NaHPO 4 , 0.25 M NaCl, 1 mM EDTA, and 100 g/ml denatured salmon sperm DNA. The membranes were washed to a final stringency of 0.25ϫ SSC and 0.2% SDS at 55-60°C for 20 -30 min. Glyceraldehyde-3-phosphate dehydrogenase mRNA was used as an internal control.
Apoptosis Analysis-Cells undergoing apoptosis were quantitated by staining with the fluorescein isothiocyanate-conjugated annexin V and the fluorescent dye propidium iodide according to the manufacturer's recommendations (Roche Molecular Biochemicals). The RA-treated cells of 6-cm dishes were harvested and incubated for 10 min at room temperature in annexin V-and propidium iodide-containing buffer and then washed with PBS. 10,000 events were analyzed per sample using a FACSCalibur-S system (BD Biosciences).
293T and SK-N-BE (2)C cells grown in sterile coverslips were transfected with pEGFP, an expression vector encoding GFP, together with expression vectors encoding the indicated proteins. In 293T cells, after 24 h of transfection, the cells were treated with TNF-␣ (20 ng/ml) and cycloheximide (10 g/ml) for 14 h. In SK-N-BE (2)C cells, after 24 h, the medium was replaced with fresh medium without FBS, containing 5 M RA, and the cells were further incubated for 2-3 days. Stimulations were terminated by aspirating the culture medium and fixing cells with ice-cold 100% methanol for 5 min at room temperature. The cells were washed three times with PBS and then stained with a BisBenzimide (H 33258) in PBS. The coverslips were washed two times with PBS, then mounted on glass slides, using Gelvatol, and visualized under a fluorescence microscope. The percentage of apoptotic cells was calculated as the number of GFP-positive cells with apoptotic nuclei divided by the total number of GFP-positive cells.
Confocal Microscopic Analysis-293T cells were grown on sterile coverslips and transfected with GFP-B-MYB and/or FLAG-tagged ZPR9 constructs by the calcium phosphate precipitation method, placed on ice, and washed three times with ice-cold PBS prior to fixation with 4% paraformaldehyde for 10 min at room temperature. The mouse monoclonal anti-FLAG (M2) antibody was applied for 2 h at 37°C. The cells were then incubated with Texas Red-conjugated anti-mouse secondary antibody (Amersham Biosciences) at 37°C for 1 h as described (31). The coverslips were washed three times with PBS and then mounted on glass slides, using Gelvatol. Confocal laser scanning microscopy observations were done on a Bio-Rad MRC 1024 (15-milliwatt argon-krypton laser; mounted on a Zeiss Axioskop (Jena, Germany) equipped with a ϫ63 (NA 140) oil-immersion objective, using a 488-(GFP) and 568-nm (Texas Red) bandpass filter).

B-MYB Physically
Interacts with ZPR9 in Vivo-We have identified recently that B-MYB proteins associate in vivo with each other (32). In an effort to identify further proteins that interact with B-MYB, we used a two-hybrid specificity test technique that was usually employed to verify the interaction specificity between bait and the cDNA-encoded proteins and to eliminate quickly the majority of false positives detected in the yeast two-hybrid assay. Specific interacting proteins confer the galactose-dependent Leu ϩ /LacZ ϩ phenotype to yeast containing the related baits but not to yeast containing unrelated baits. To test this, the B-MYB library plasmid was rescued from the galactose-dependent Leu ϩ /LacZ ϩ yeast and re-introduced into the ZPR9 bait strain as well as the other strains containing approximately 20 different baits available in our laboratory. From this random screening, B-MYB cDNA was found to interact with the total seven baits tested (results not shown), including ZPR9 and B-MYB baits, suggesting that ZPR9, like B-MYB (32), can interact with B-MYB physically in mammalian cells.
To determine whether B-MYB and ZPR9 interact in vivo, we performed cotransfection experiments using GST-and FLAGtagged eukaryotic expression vectors. In these experiments, the ZPR9 and wild-type B-MYB were coexpressed as a GST fusion protein and a FLAG-tagged protein in 293T cells, respectively. The interactions of FLAG-tagged B-MYB proteins to the GST-ZPR9 fusion proteins were analyzed by immunoblotting with an anti-FLAG antibody. As shown in Fig. 1A, the B-MYB was detected in the coprecipitate only when coexpressed with the GST-ZPR9 but not with the control GST alone, demonstrating that B-MYB physically interacts with ZPR9 in vivo. In order to verify further the interaction of B-MYB with ZPR9 in vivo, we performed coimmunoprecipitation experiments using 293T cells transiently transfected with the vector alone or FLAG-tagged ZPR9 (Fig. 1B). Endogenous B-MYB was immunoprecipitated from cell lysates, and Western blot analysis shows that B-MYB was precipitated (Fig. 1B, lower panel). The binding of ZPR9 was subsequently analyzed using Western blotting with an anti-FLAG antibody, and as shown in Fig. 1B  (upper left panel), ZPR9 was present in the B-MYB immunoprecipitate. In conclusion, our results clearly demonstrate that B-MYB associates with ZPR9 in vivo.
Three Functional Domains of B-MYB Are Involved in ZPR9 Binding-Recently, together with other data (22,23), it was reported that poly(ADP-ribose) polymerase binds to the B-MYB DNA-binding domain and enhances the transcriptional activity of B-MYB (21). Therefore, we speculated that ZPR9 might interact with the DNA-binding or transactivation domain of B-MYB, in addition to the carboxyl-terminal conserved region, and cause the modulation of B-MYB transactivation. To determine which regions of B-MYB were required for binding of ZPR9 in vivo, we generated nine deletion constructs fused to GST (Fig. 2, A and B). The GST-WT B-MYB, GST-CR, GST-B-MYB, GST-TA1, GST-B-MYB R1, GST-TA, and GST-B-MYB R2 constructs were expressed in 293T cells (Fig. 2, C and D, middle left panels) and used for the in vivo binding assay with ZPR9 and Two9, a partial clone of ZPR9 comprising amino acids 206 -452 (31). The binding of the FLAG-tagged ZPR9 and Two9 with all six constructs tested, except for GST-B-MYB R2, was readily detectable (Fig. 2, C and D, top left panels). These results suggest that all three functional domains of B-MYB, a DNA-binding domain, a transactivation domain, and the carboxyl-terminal conserved region, are responsible for ZPR9 binding in vivo. To narrow down further the binding motif, we generated a DBD deletion construct (amino acids 1-206) and carried out a similar experiment. As a result, the FLAG-tagged DBD was coprecipitated with GST-tagged ZPR9 (or Two9) but not with GST alone (Fig. 2, C and D, top right panels). These findings, together with the binding of ZPR9 to GST-TA, clearly indicate that both DNA-binding and transactivation domains are required for ZPR9 binding. However, the GST-B-MYB R2 was not coprecipitated with FLAG-tagged ZPR9 or Two9, indicating that the conserved region is only required for ZPR9 binding within the carboxyl-terminal domain of B-MYB. Taken together, these results suggest that each functional domain of B-MYB is sufficient for its association with ZPR9.
ZPR9 Enhances the Transcriptional Activity of B-MYB-Because ZPR9 is binding to the DNA-binding and transactivation domain of B-MYB (Fig. 2), it is likely that the interaction may affect the transactivation by B-MYB. To investigate the functional significance of binding of ZPR9 to B-MYB, we cotransfected the pT81luc 3xA reporter plasmid, containing three Myb-binding sites from the chicken mim-1 gene (33) ZF2, and ZF3). Amino acid number of domain boundaries is indicated. C and D, mapping of the site on B-MYB involved in the association with ZPR9 and Two9. 293T cells were cotransfected with GST alone (pEBG) or the deletion mutants as indicated, together with pFLAG-ZPR9 (FLAG-ZPR9) or pFLAG-Two9 (FLAG-Two9). Transfected cells were extracted and purified with glutathione-Sepharose beads (GST purification) and immunoblotted with an anti-FLAG antibody as in Fig. 1. A complex formation between B-MYB proteins and ZPR9 (or Two9) was determined by Western analysis (WB) using anti-FLAG antibody (top panels). The same blot was re-probed with an anti-GST antibody to examine the expression of GST fusion proteins in the coprecipitates (middle panels), and the expression level of FLAG-tagged proteins in total cell lysates (Lysate) was analyzed by Western analysis using anti-FLAG antibody (bottom panels). GST alone (pEBG) and either pEBG-ZPR9 (GST-ZPR9) or pEBG-Two9 (GST-Two9) were cotransfected with pFLAG-DBD (FLAG-DBD) into 293T cells. Transfected cells were extracted and purified with glutathione-Sepharose beads and immunoblotted with an anti-FLAG antibody to analyze a complex formation (top right panels). The expression level of FLAG-tagged and GST fusion proteins in total cell lysates and the coprecipitates was analyzed by anti-FLAG (bottom right panels) and anti-GST (middle right panels) immunoblot assays. The asterisks indicate the expressed GST fusion proteins.
B-MYB and ZPR9 exhibit both cytosolic and nuclear staining, but the coexpression of ZPR9 with B-MYB resulted in nuclear localization of the B-MYB protein, as well as the ZPR9, with an average increase of 2.8-and 3.8-fold in the four experiments, respectively, when the percentage of the nuclear localization of B-MYB and ZPR9 was calculated as the number of GFP-(for B-MYB) and Texas Red-positive (for ZPR9) cells with nuclear staining divided by the total number of GFP-and Texas Redpositive cells (Fig. 4). These data show that ZPR9 is able to cooperate with B-MYB for the transactivation by B-MYB.
Constitutive Expression of ZPR9 Induces Apoptotic Neuroblastoma Cell Death by Retinoic Acid-To analyze the effect of ZPR9 gene expression on the differentiable or apoptotic potential of SK-N-BE (2)C, a human neuroblastoma cell line, we constructed an expression vector pCEV27-ZPR9, where a fulllength human ZPR9 cDNA was placed under the control of Moloney murine leukemia virus long terminal repeat promoter. ZPR9-transfected SK-N-BE (2)C cells were selected in medium containing G418 (800 g/ml). Overexpression of the ZPR9 transcript in the selected transfectants was analyzed by Northern blot analysis. As shown in Fig. 5A, compared with parental SK-N-BE (2)C cells and pCEV27 vector transfectants, as negative controls, the ZPR9 transcripts were identified at a high level in the selected ZPR9 transfected clones. In addition, similar results were obtained with all selected ZPR9 transfectant clones (results not shown). The growth rates under normal serum conditions were comparable in ZPR9 transfectants, pCEV27 vector transfectants, and parental SK-N-BE (2)C cells, suggesting that the ectopic expression of ZPR9 did not affect the proliferative activity on neuroblastoma cells (results not shown). RA treatment resulted in a more rapid loss of viability in all ZPR9-expressing clones compared with the parental SK-N-BE (2)C cells and the cell lines transfected with the pCEV27 vector (Fig. 5B). To confirm if the marked decrease in cellular viability of the RA-treated ZPR9 transfectants is due to apoptosis, we performed dual annexin V/propidium iodide staining as described under "Experimental Procedures" and obtained an experimental result similar to those in Fig. 5B. A significant increase in the number of apoptotic cells was observed in the ZPR9 transfectants after RA treatment, suggesting that the  Forty eight hours after transfection, cells were washed three times with ice-cold PBS and fixed with 4% paraformaldehyde for 10 min at room temperature prior to incubation with the anti-FLAG (M2) monoclonal antibody, which was followed by an incubation with a Texas Redconjugated anti-mouse secondary antibody to label the ZPR9 construct. Slides were mounted and analyzed by confocal microscopy. B, effect of B-MYB on ZPR9 subcellular localization. 293T cells were transfected with FLAG-tagged ZPR9 alone (ZPR9), as a control, or GFP-B-MYB was coexpressed in cells together with FLAG-tagged ZPR9 (ZPR9/B-MYB). Cells were washed and fixed as described above. Cells were immunostained with the anti-FLAG (M2) monoclonal antibody, followed by Texas Red-conjugated anti-mouse secondary antibody, and analyzed by confocal microscopy. GFP is in green, and areas of colocalization appear as yellow (Merge).
overexpression of ZPR9 may induce apoptosis, instead of the neural differentiation, in the presence of RA (Fig. 5C). To investigate further the physiological roles of ZPR9 during apoptosis, 293T cells were transiently transfected with GFP alone, GFP and ZPR9, and GFP and B-MYB. In addition, cells were cotransfected with ZPR9 and B-MYB, together with GFP. After inducing apoptosis by TNF-␣ treatment, apoptotic cells were scored by a change in nuclear morphology among GFP-positive FIG. 5. ZPR9 transfectants undergo apoptotic cell death after RA treatment. A, SK-N-BE (2)C cells were stably transfected with a ZPR9 expression plasmid (pCEV27-ZPR9) or the parental plasmid (pCEV27) as a control. The relative expression levels of ZPR9 mRNA in G418-resistant clones were examined by Northern blot analysis. Total cytoplasmic RNAs were extracted from ZPR9 transfectants (ZPR9 -18, -34, -37, and -38), pCEV27 transfectants (V11 and V16), and parental SK-N-BE (2)C cells (SK), and the membrane was probed with a 32 P-labeled ZPR9 cDNA insert. Exo-ZPR9 represents the exogenous ZPR9 mRNA, and endo-ZPR9 represents the endogenous ZPR9 mRNA. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. B, cells were seeded in 6-well plates at 4 ϫ 10 5 cells per well the day before RA treatment. The cell number of viable cells at the indicated times following RA treatment of cells (SK, V16, ZPR9 -34, and ZPR9 -37), determined by trypan blue exclusion, was counted with a hemocytometer. Each point indicates the means Ϯ S.E. of two separate experiments carried out in triplicate. C, cells were plated at the density of 3-4 ϫ 10 5 cells/cm 2 and grown the day before RA treatment in the presence of 10% serum. Serum starvation was induced by changing the medium to 0% serum and then cultured in the presence (shaded bars) or absence (white bars) of RA for 2 days. Apoptotic cell death was determined by flow cytometry for annexin V and propidium iodide. Results shown are the average of duplicate samples and are representative of two independent experiments. D, 293T cells were transiently transfected with expression vectors encoding B-MYB (5 g) and ZPR9 (5 g) along with an expression vector encoding GFP (3 g) as indicated. Transfected cells were incubated for 24 h and treated with TNF-␣ (20 ng/ml) and cycloheximide (10 g/ml) for 14 h to induce apoptosis. E, SK-N-BE (2)C cells were transiently transfected with an expression vector encoding GFP and the indicated combinations of expression vectors encoding B-MYB and ZPR9. After 24 h, the medium was changed with 0% serum, and then cells were cultured in the presence of RA for 2 or 3 days to induce apoptosis. D and E, GFP-positive cells were analyzed for the presence of apoptotic nuclei with a fluorescence microscope. The data shown are the mean Ϯ S.D. of duplicate assays and are representative of at least three independent experiments. cells. As shown in Fig. 5D, ϳ59% of 293T cells expressing ZPR9 were apoptotic following TNF-␣ treatment. In contrast, ϳ16% of cells transfected with B-MYB underwent TNF-␣-induced apoptosis, similar to the percentage (about 15%) of control apoptotic cells expressing GFP alone. On the other hand, B-MYB coexpression markedly inhibited the apoptotic stimulation induced by ZPR9 (about 35% inhibition).
To confirm further the involvement of ZPR9 in the enhancement of RA-induced apoptosis, we carried out a similar transient transfection experiment using SK-N-BE (2)C cells. As shown in Fig. 5E, the results obtained in this experiment were very similar to those in Fig. 5D. These findings suggest that the overexpression of ZPR9 is sufficient to stimulate apoptosis induced by various stimuli and raise the possibility that ZPR9 may be a potential pro-apoptotic protein. DISCUSSION In this report, we demonstrate that ZPR9 interacts with B-MYB in vivo and that each functional domain of B-MYB is necessary and sufficient to mediate direct protein-protein interactions with ZPR9. We found that ZPR9 enhanced the transactivating activity of B-MYB by direct interaction. Furthermore, we show that B-MYB moves to the nucleus following the coexpression of ZPR9, implying that ZPR9 may behave as an activator of the bound transcription factor, B-MYB.
The novel zinc finger protein, termed ZPR9 (zinc finger-like protein 9), was originally discovered as a protein partner for the MPK38 serine/threonine kinase (31). Recently, evidence has emerged that several zinc finger proteins such as ZPR1, tumor necrosis factor receptor-associated factor, CD40 receptor-associated factor, enigma, and LMP-associated protein act as modulators for receptor signaling, and that the formation of multiprotein complexes in many transcription factors results in an increased diversity and specificity in the regulation of gene expression (35)(36)(37)(38)(39)(40). Zinc finger motifs of the Cys 2 -His 2 type have been found in numerous transcription factors, including ZPR9. In this respect, the self-association of ZPR9 containing zinc finger motifs and the interaction of ZPR9 with the kinase catalytic domain of MPK38 provide an interesting aspect to the regulation of this factor (31). In addition, our recent study strongly suggests a possible role for phosphorylation of ZPR9 proteins in their translocation to the nucleus (31). Thus, these data open a new area of investigation on the potential interaction of ZPR9 with other cellular proteins.
Several lines of evidence indicate that the DNA-binding domain of MYB proteins has the potential of mediating contact with both DNA and proteins. It has been shown that PARP binds to the DNA-binding domain of B-MYB and enhances its transactivating activity and that the physical interaction between PARP and B-MYB is critical for the coactivating function (21), suggesting an important role for the direct interaction in the regulation of the B-MYB transcriptional activity. Recently, we have shown that B-MYB interacts in vivo with each other via the carboxyl-terminal conserved region (32). In addition, we have observed that the conserved region of B-MYB binds to several cellular proteins as well as ZPR9 in the yeast twohybrid tests and in vivo binding assays (results not shown). This evidence led us to investigate whether ZPR9, a potential transcription factor containing zinc finger motifs, participates in the B-MYB-mediated transactivation. As shown in Fig. 3, a significant increase was observed in the transactivating activity of B-MYB by direct binding of ZPR9, suggesting that the in vivo association of B-MYB and ZPR9 plays a pivotal role in the modulation of B-MYB transcriptional activity. Based on this result, we imagine that rather than direct interaction with the carboxyl-terminal conserved region, the conformational change mediated through the DNA-binding or transactivation domain of B-MYB by direct binding of ZPR9 likely plays a role that is important in the regulation of B-MYB transcriptional activity. For activation of B-MYB transcriptional activity, our results, together with existing data (21)(22)(23), suggest a distinct mechanism in which, in addition to the truncation of the carboxyl terminus of B-MYB and the phosphorylation by cyclin A-CDK2 complex (7)(8)(9)(10), cellular cofactors are bound to the B-MYB, for example through the binding of PARP and ZPR9, resulting in the enhancement of the B-MYB transcriptional activity (21,31).
Recent studies (41) showed that all-trans-retinoic acid reduces human neuroblastoma growth by inducing either differentiation or apoptosis. To determine whether RA-treated ZPR9 stable transfectants can influence the differentiation or apoptosis in human neuroblastoma cells, the morphological and apoptotic analysis of ZPR9 transfectants was performed in addition to the examination of the number of viable cells ( Fig.  5 and results not shown). The ZPR9 transfectants undergo apoptosis rather than differentiation after RA treatment, suggesting that ZPR9 may be one of the regulators controlling cellular growth arrest induced by RA in neuroblastoma cells. To test whether the observed apoptotic cell death after RA treatment in the ZPR9 stable transfectants is a consequence of direct interaction of B-MYB with ZPR9, we performed two separate transient transfection experiments using 293T and SK-N-BE (2)C cells in the presence of TNF-␣ and RA, respectively (Fig. 5, D and E). In these experiments, coexpression of B-MYB significantly inhibited rather than stimulated TNF-␣ and RA-induced apoptosis enhanced by ZPR9. In contrast, the percentage of apoptosis in cells transfected with B-MYB alone was similar to the percentage of control apoptotic cells expressing GFP alone in both 293T and SK-N-BE (2)C cells. Thus, it is tempting to suggest that the apoptotic cell death in RA-treated ZPR9 stable transfectants may be derived from the overexpression of ZPR9 itself, not through binding with B-MYB. On the other hand, one may raise the argument that ZPR9, like other zinc finger proteins, could be nuclear for its effect on target genes. Based on this, one possible explanation for ZPR9-induced apoptosis is that a subcellular location of ZPR9 may contribute to its apoptotic function in the presence of retinoic acid because a markedly increased nuclear accumulation of ZPR9, compared with the untreated control cells, was observed when the cells transfected with ZPR9 were treated with RA (results not shown). Additionally, it is not clear that the repression of ZPR9-induced apoptosis by B-MYB is dependent on the direct interaction of B-MYB with ZPR9 because B-MYB thought to mediate the anti-apoptotic functions is also involved in interactions with other cellular proteins that may compete with ZPR9 for binding (see Refs. 21 and 32 and results not shown). The biochemical and molecular mechanisms underlying the pro-apoptotic properties of ZPR9 are unknown at present. However, it seems that the most likely mechanism by which ZPR9 may accelerate apoptosis would be through the modulation of the potential cellular targets for ZPR9. In this context, future studies aimed at identifying cellular physiological targets for ZPR9 will be necessary to elucidate the exact mechanism through which ZPR9 can induce apoptotic cell death.
In addition, ZPR9 proteins, like PARP (21), are nuclear and enhance B-MYB transactivation. In this regard, it will be of further interest to determine the mechanistic interaction between B-MYB and ZPR9 to gain more insight into the role of ZPR9 in the B-MYB transactivation. Moreover, because B-MYB is thought to have a general role in cell growth control, differentiation, and cancer, the ZPR9-dependent modulation of this transcription factor may contribute to elucidate the mech-anism by which B-MYB affects these processes as well as the mechanism of transcription activation by B-MYB.